Chapter 4 Discussion
4.6 Outlook
Given the recent advances in the field of single-molecule manipulation, the Nb-GFP complex offers great opportunities for further expansion, e.g. in the cut-and-paste technology [Kufer et al., 2008]. Direct follow-up experiments at this point could include identifying three different types of Nanobodies suited as handles to immo-bilize, pick up and deposit elsewhere GFP-tagged proteins. Based on the findings presented in [Kirchhofer et al., 2010], one of the two steps of this GFP takeover could be realized by the pair referred to as Minimizer and Enhancer in the abovementioned publication, since these two binders display such a hierarchical behavior. Thanks to the popularity of the GFP-tag, most proteins have already been fused to the GFP, which makes this strategy straightforward and ready to use once such a GFP-binder based system has been established.
Appendix A
List of abbreviations
◦ 3D - 3-dimensional
◦ 5hmC - 5-hydroxymethylcytosine
◦ Å - Ångstrom
◦ AFM - Atomic Force Microscope
◦ bp - basepair
◦ BG - benzylguanine
◦ CDR - Complementarity Determining Region
◦ CE - coupling efficiency
◦ CoA - Coenzyme A
◦ Co-IP - Co-Immunoprecipitation
◦ CpG - C-phosphate-G
◦ dCTP - deoxycytidine triphosphate
◦ ddH2O - double-distilled water
◦ DMF - dimethylformamide
◦ DNA - deoxyribonucleic acid
◦ dsDNA - double-stranded deoxyribonucleic acid
◦ dNTP - deoxynucleotide
◦ EDC - 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
106 A. List of abbreviations
◦ dUTP - deoxyuridine triphosphate
◦ eGFP - enhanced Green Fluorescence Protein
◦ EMSA - Electrophoretic Mobility Shift Assay
◦ Fab - Fragment, antigen-binding
◦ FJC - Freely Jointed Chain
◦ fN - femtonewton
◦ FRET - Fluorescence Resonance Energy Transfer
◦ GA - Gibson Assembly
◦ GFP - Green Fluorescence Protein
◦ h - hour
◦ hAGT - human O6-alkylguanine-DNA alkyl transferase
◦ HCAb - heavy-chain-only antibody
◦ HisTag - polyhistidine-tag
◦ Ig - immunoglobulin
◦ kB - Boltzmann constant
◦ Kd - dissociation constant
◦ kb - kilobase
◦ kDa - kilodalton
◦ LB - lysogeny broth
◦ MBD - Methyl-DNA Binding Domain
◦ MeCP2- methyl CpG binding protein 2
◦ MFA - Molecular Force Assay
◦ MFB - Molecular Force Balance
◦ min - minute
◦ mm - millimeter
◦ mM - millimolar
◦ MT - Magnetic Tweezers
◦ µl - microliter
◦ µM - micromolar
107
◦ NA - Neutravidin
◦ Nb - Nanobody
◦ NF - normalization factor
◦ NHS - N-hydroxysuccinimide
◦ nm - nanometer
◦ NMR - Nuclear Magnetic Resonance
◦ PAGE - Polyacrylamide Gel Electrophoresis
◦ PBS - Phosphate-buffered saline
◦ PCR - Polymerase Chain Reaction
◦ PDMS - polydimethylsiloxane
◦ PEG - Polyethylene Glycol
◦ PMSF - phenylmethanesulfonyl fluoride
◦ pN - piconewton
◦ RNA - Ribonucleic acid
◦ RTT - Rett Syndrome
◦ s - second
◦ SA - streptavidin
◦ sfGFP - superfolder Green Fluorescence Protein
◦ SFP - 4´-phosphopantetheinyl transferase
◦ SM - Single-Molecule
◦ TCEP - tris(2-carboxyethyl)phosphine
◦ UHRF1 - Ubiquitin-like containing PHD and RING finger domains 1
◦ UV - ultraviolet
◦ v/v - volume/volume
◦ WDR - WW domain-binding region
◦ WLC - Wormlike Chain
◦ wtGFP - wild-type Green Fluorescence Protein
◦ Y2H - Yeast Two-Hybrid
Appendix B
Declaration
Kamila Klamecka
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